The displacement mode of chromatography was first recognized in 1906 by Tswett, who noted that sample displacement occurred under conditions of overloaded elution chromatography. In 1943, Tiselius proposed that the broad subject of chromatography could be organized around three distinct “modes:” frontal mode, elution mode, and displacement mode. Since then, most developments and applications, particularly those in analytical chromatography, have taken place in the area of elution chromatography. Indeed, the term “chromatography” without further qualification today usually refers to chromatography in the elution mode.
Elution mode and displacement mode are readily distinguished both in theory and in practice. In elution chromatography, a solution of the sample to be purified is applied to a stationary phase, commonly in a column. The mobile phase is chosen such that the sample is neither irreversibly adsorbed nor totally non-adsorbed, but rather binds reversibly. As the mobile phase is flowed over the stationary phase, an equilibrium is established between the mobile phase and the stationary phase whereby, depending upon the affinity for the stationary phase, the sample passes along the column at a speed which reflects its affinity relative to other components that may be present in the original sample. Of particular note in standard elution chromatography is the fact that the eluting solvent front, or zero column volume in isocratic elution, always precedes the sample off the column.
A modification and extension of isocratic elution chromatography is found in step gradient chromatography wherein a series of eluents of varying composition are passed over the stationary phase. In ion-exchange chromatography, for example, step changes in the mobile phase salt concentration and/or pH are employed to elute or desorb analytes undergoing separation.
Displacement chromatography employs a displacer compound to remove components of a mixture from the column. The displacer compound generally has a much higher affinity for the stationary phase than do any of the components in the mixture to be separated. This is in contrast to elution chromatography, where the eluent has a lower affinity for the stationary phase than do the components to be separated. A key operational feature that distinguishes displacement chromatography from elution or desorption chromatography is the use of a displacer compound. In displacement chromatography, the column is first equilibrated with a carrier solvent under conditions in which the components to be separated all have a relatively high affinity for the stationary phase. A volume of feed mixture, which can be large and quite dilute, is loaded onto the column and individual components in the feed mixture will adsorb to the stationary phase. That is, the components of the feed mixture are distributed and adsorbed onto the stationary phase, and remain there. If all the components are to be resolved by displacement, the carrier solvent emerges from the column containing no sample. The components of the feed mixture now reside on the stationary phase, and the position of each component on the column is correlated with its relative affinity for the stationary phase under the initial conditions. In principle, a molecule of any component will displace a molecule of any different component having a lower affinity at a given site on the stationary phase. As a result, individual components will ultimately be arranged on the column in sequence from highest to lowest affinity.
It is sometimes advantageous to allow some components of the feed mixture, e.g., components not having a significant affinity for the stationary phase, to pass through the column with the carrier solvent; in this case only the retained feed components will be resolved by displacement chromatography.
Once the sample is loaded on the column, a solution containing a displacer compound in a suitable solvent is introduced into the column to pass through the stationary phase. The displacer compound is selected such that it has a higher affinity for the stationary phase than do any of the components of the feed mixture. Assuming that the displacer and mobile phase are appropriately chosen, the individual components exit the column as adjacent zones of highly concentrated, relatively pure material in the order of increasing affinity of absorption. Following the zones of the purified individual components, the displacer emerges from the column. A displacement chromatogram is readily distinguished from an elution chromatogram by virtue of the fact that the displacer compound follows the sample and that the feed components exit the column as adjacent zones of highly concentrated, relatively pure material.
Displacement chromatography has some particularly advantageous characteristics for process scale chromatography. First, displacement chromatography can achieve product separation and concentration in a single step. By comparison, isocratic elution chromatography results in significant product dilution during separation. Second, since the displacement process operates in the nonlinear region of the equilibrium isotherm, high column loadings are possible. This allows much better column utilization than elution chromatography. Third, column development and component separation requires less solvent than a comparable elution process. Fourth, displacement chromatography can concentrate and purify components from mixtures having low separation factors, while relatively large separation factors are required for satisfactory resolution in typical elution chromatography.
With all of these advantages, one might presume that displacement chromatography would be widely utilized. However, problems have persisted in displacement chromatography. One such problem is the need to regenerate the column, since it would not be economical to discard the stationary phase after each use. Another such problem is obtaining suitable displacer compounds that are relatively simple compounds, easily synthesized and/or commercially available at a reasonable (economical) cost. These two problems have presented significant drawbacks to displacement chromatography vis-a-vis elution chromatography.
With respect to regeneration, since the displacement process uses a displacer compound having a very high affinity for the stationary phase, the time needed to regenerate and re-equilibrate the column can be long compared to elution chromatography. Furthermore, relatively large amounts of solvent are often required during regeneration. These problems have effectively reduced the advantages of displacement chromatography over elution chromatography.
The second problem, that of obtaining useful displacer compounds that can be synthesized relatively easily and/or that are commercially available at a reasonable (economical) cost, is due to the need for a displacer compound that has both a high affinity for the stationary phase but that can also be relatively easily removed from the column during regeneration. Such compounds that have been offered by the prior art do not meet one or both of these two important criteria. Various compounds have been offered as low molecular weight displacers, but these have been quite difficult to synthesize and purify and have not been commercially available at reasonable cost, or simply not commercially available.
In order for displacement chromatography to become a mainstream chromatographic technique, there remains a significant unmet need for effective displacers whose synthesis and purification are straightforward and that are amenable to large-scale production, and/or that are commercially available, and which allow for efficient regeneration of the stationary phase so that the stationary phase can be subsequently reused in displacement chromatography processes.